Giovanna Pulcrano

MALDI-TOF mass spectrometry and microsatellite markers to evaluate Candida parapsilosis transmission in neonatal intensive care units.

Recent studies on outbreaks of Candida showed an increased incidence of bloodstream infections in neonatal intensive care units (NICUs) caused by C. parapsilosis species, highlighting the need for the proper identification and epidemiology of these species. Several systems are available for molecular epidemiological and taxonomic studies of fungal infections: pulsed-field gel electrophoresis (PFGE) represents the gold standard for typing, but is also one of the most lengthy and expensive, while simple sequence repeats (SSRs) is based on polymerase chain reaction (PCR) amplification and is, therefore, faster. Only recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify and type microorganisms involved in nosocomial outbreaks. In our study, 19 strains of C. parapsilosis isolated from the blood cultures of neonates admitted to the University Hospital Federico II were genotyped by the amplification of eight SSR markers and by MALDI-TOF MS. Electrophoretic and spectrometric profile results were compared in order to identify similarities among the isolates and to study microevolutionary changes in the C. parapsilosis population. The discriminatory power and the unweighted pair group method with arithmetic mean (UPGMA) dendrograms generated were compared in order to evaluate the correlation of the groups established by the analysis of the clusters by both methods. Both methods were rapid and effective in highlighting identical strains and studying microevolutionary changes in the population. Our study evidenced that mass spectroscopy is a useful technique not only for the identification but also for monitoring the spread of strains, which is critical to control nosocomial infections.

Rapid and reliable MALDI-TOF mass spectrometry identification of Candida non-albicans isolates from bloodstream infections

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has recently become an effective instrument for rapid microbiological diagnostics and in particular for identification of micro-organisms directly in a positive blood culture. The aim of the study was to evaluate a collection of 82 stored yeast isolates from bloodstream infection, by MALDI-TOF MS; 21 isolates were identified also directly from positive blood cultures and in the presence of other co-infecting micro-organisms. Of the 82 isolates grown on plates, 64 (76%) were correctly identified by the Vitek II system and 82 (100%) by MALDI-TOF MS; when the two methods gave different results, the isolate was identified by PCR. MALDI-TOF MS was unreliable in identifying two isolates (Candida glabrata and Candida parapsilosis) directly from blood culture; however, direct analysis from positive blood culture samples was fast and effective for the identification of yeast, which is of great importance for early and adequate treatment.

Typing of Ochrobactrum anthropi clinical isolates using automated repetitive extragenic palindromic-polymerase chain reaction DNA fingerprinting and matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry

BackgroundOchrobactrum anthropi (O. anthropi), is a non-fermenting gram-negative bacillus usually found in the environment. Nevertheless, during the past decade it has been identified as pathogenic to immunocompromised patients. In this study, we assessed the usefulness of the automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR-based DiversiLab™ system, bioMèrieux, France) and of matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF MS) for typing of twentythree O. anthropi clinical isolates that we found over a four-months period (from April 2011 to August 2011) in bacteriemic patients admitted in the same operative unit of our hospital. Pulsed-field gel electrophoresis (PFGE), commonly accepted as the gold standard technique for typing, was also used. Analysis was carried out using the Pearson correlation coefficient to determine the distance matrice and the unweighted pair group method with arithmetic mean (UPGMA) to generate dendogram.ResultsRep-PCR analysis identified four different patterns: three that clustered together with 97% or more pattern similarity, and one whose members showed < 95% pattern similarity. Interestingly, strains isolated later (from 11/06/2011 to 24/08/2011) displayed a pattern with 99% similarity. MALDI-TOF MS evaluation clustered the twentythree strains of O. anthropi into a single group containing four distinct subgroups, each comprising the majority of strains clustering below 5 distance levels, indicating a high similarity between the isolates.ConclusionsOur results indicate that these isolates are clonally-related and the methods used afforded a valuable contribution to the epidemiology, prevention and control of the infections caused by this pathogen.

Clonal dissemination of Klebsiella pneumoniae ST512 carrying blaKPC‐3 in a hospital in southern Italy

Strains of Klebsiella pneumoniae producing KPC‐carbapenemase have emerged as one of the most important multidrug‐resistant Gram‐negative nosocomial pathogens. Here, we report the first isolation and subsequent dissemination of a K. pneumoniae ST512 producing KPC‐3 carbapenemase in a hospital in southern Italy. Isolates were obtained from blood, throat swabs, sputum, catheters, and urine of patients admitted to different hospital wards. Antimicrobial MICs were determined for all isolates by automated systems and confirmed by Etest. Carbapenemase production was confirmed by the modified Hodge test and by a disc synergy test, and carbapenemase genes were investigated by PCR. All isolates were characterized by pulse‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analysis. Most isolates were multidrug resistant with exception of some isolates intermediately susceptible to gentamicin, tigecycline, and trimethoprim‐sulfamethoxazole. PCR analysis showed that isolates harbored the blaKPC‐3 gene associated with blaTEM and blaSVH. PFGE and MLST showed that all isolates belonged to the same ST512 clone recently described in Israel.

Ultrasmall silver nanoparticles loaded in alginate–hyaluronic acid hybrid hydrogels for treating infected wounds

ABSTRACT Nowadays, silver nanoparticles are in the limelight to control infection during wound healing process, and a vast variety of antimicrobial dressings based on colloidal silver have been marketed to fight wound invasion of pathogen bacteria, which represents one of the main adverse effects limiting the repair process. Here we propose a biofunctional hydrogel based on alginate (ALG) and hyaluronic acid (HA) embedding ultrasmall silver nanoparticles (usSN, <1 nm) as antimicrobial component. The hydrogels were fabricated in different size by a straightforward internal gelation method using CaCO3 and glucono-δ-lactone. To follow usSN release from the hydrogels in aqueous media, catalytic activity of usSN-loaded hydrogels was evaluated. Results suggested that catalytic activity was low in intact hydrogels and high when hydrogels dissolved, which suggests that usSN firmly interact with polymer chains and are available in the medium depending on the extent of hydrogel degradation. HA-containing hydrogels showed faster dissolution in simulated physiological conditions and higher antibiofouling properties as compared to hydrogels made only of ALG. Free usSN were not toxic toward human mesenchymal stem cells (Ad-MSCs), previously isolated from subcutaneous adipose tissue biopsies, up to 50 µg/mL. At this concentration, viability of Ad-MSCs was unaffected whereas their motility was significantly higher as compared to control (p<0.01) for both free usSN and hydrogel integrating. Antimicrobial activity on clinical isolates of both Gram-positive and Gram-negative bacteria demonstrated that usSN at 50 µg/mL were able to kill all the bacteria tested after 24 and 48 h of contact time. In the case of hydrogels, a matrix effect was found and bacterial killing was significant only at 24 h and dependent on bacterial strain, being Gram-negative bacteria more susceptible. These results clearly indicate that usSN interaction with polymer network and exposure time can strongly affect usSN antimicrobial profile in the hydrogel and, in turn, timing of hydrogel change at injured site in a clinical setting. On the whole, ALG/HA hydrogels integrating usSN can be considered a suitable option to fabricate biofunctional dressings for hospitalized patients and worth of further in vivo investigation. GRAPHICAL ABSTRACT

In vitro antimicrobial profile of Ureaplasma urealyticum from genital tract of childbearing-aged women in Northern and Southern Italy.

Ureaplasma urealyticum is an opportunistic pathogen during pregnancy and in newborns. Other clinical problems related to U. urealyticum infections are: no susceptibility to cell wall‐active drugs, limits of antibiotic treatment in pregnancy, and spread of antimicrobial resistance. In addition, the results of antimicrobial susceptibility against U. urealyticum from various countries are few and controversial. The antimicrobial susceptibility of U. urealyticum, isolated from cervical swabs and collected from outpatient childbearing‐aged women in Italy from 2009 to 2012, was performed against fluoroquinolones, macrolides, streptogramin and tetracyclines, using an available biochemical commercial kit and a specific solid culture medium, to improve the therapeutic management of these pathogenic agents. Ureaplasma urealyticum was detected in 49.4% of samples, but significant bacterial load was revealed in 29.8%. In vitro tetracyclines showed the best activity against U. urealyticum, followed by streptogramin, macrolides, and fluoroquinolones.

Molecular and phenotypic characterization of methicillin-resistant Staphylococcus aureus from surgical site infections.

BACKGROUND Nosocomial infections represent an important problem for the health of hospitalized patients. Peri-operative infections--those occurring during surgery or in the post-operative period--account for 15%-20% of cases. Most surgical site infections (SSIs) are caused by endogenous gram-positive microorganisms, in particular, Staphylococcus aureus, S. epidermidis, and other coagulase-negative staphylococci that are part of the flora of the skin. METHODS A retrospective study was conducted from January 2006 to December 2010 to describe the epidemiology of methicillin-resistant S. aureus (MRSA) in SSIs. The MRSA isolates were analyzed by a combination of two genotyping methods: SCCmec and pulsed-field gel electrophoresis (PFGE). Also, biofilm-forming ability was analyzed for all isolates as an indicator of their ability to persist despite antibiotic treatment. RESULTS During the study period, 1,793 swabs from SSIs were analyzed, and S. aureus was identified in 318/987 positive specimens (32%). Methicillin resistance was observed in 10% of the S. aureus isolates (n=33). Analysis by PFGE revealed that isolates with the same SCCmec type were unrelated. Instead, biofilm-forming ability tests showed that SCCmec type I MRSA had the highest ability to form a film. CONCLUSIONS The strains analyzed in our study showed a homogeneous pattern of SCCmec type. The difference in ability to produce biofilm between strains of SCCmec type I and isolates with other SCCmecs was substantial. This virulence factor could have critical implications for the formation and persistence of chronic SSIs.

The replacement H3.3 histone gene in Paracentrotus lividus sea urchin: structure and regulatory elements.

We have isolated the Paracentrotus lividus sea urchin H3.3 histone gene and characterized the nucleotide sequences of the gene and its proximal promoter. Band shift experiments showed that two cAMP/PMA responsive elements (CRE/TRE), present in the proximal promoter, bind nuclear factors present in embryos at the blastula and gastrula stages (CRE1) and at the blastula stage (CRE2). The putative H3.3 coding region activating sequences (CRAS) failed to bind nuclear factors while the corresponding elements of the two replication-dependent genes (H3L and late H3) clearly recognized nuclear proteins. These results suggest some role of the CRE/TRE elements but not CRAS elements in the transcriptional regulation of the replication-independent histone genes in invertebrates.

Isolation of Enterobacter aerogenes carrying blaTEM‐1 and blaKPC‐3 genes recovered from a hospital Intensive Care Unit

Enterobacter aerogenes has recently emerged as an important hospital pathogen. In this study, we showed the emergence of E. aerogenes isolates carrying the blaKPC gene in patients colonized by carbapenem‐resistant Klebsiella pneumoniae strains. Two multiresistant E. aerogenes isolates were recovered from bronchial aspirates of two patients hospitalized in the Intensive Care Unit at the “Santa Maria della Scaletta” Hospital, Imola. The antimicrobial susceptibility test showed the high resistance to carbapenems and double‐disk synergy test confirmed the phenotype of KPC and AmpC production. Other investigation revealed that ESBL and blaKPC genes were carried on the conjugative pKpQIL plasmid. This is a relevant report in Italy that describes a nosocomial infection due to the production of KPC beta‐lactamases by an E. aerogenes isolate in patients previously colonized by K. pneumoniae carbapenem‐resistant. In conclusion, its necessary a continuous monitoring of multidrug‐resistant strains for the detection of any KPC‐producing bacteria that could expand the circulation of carbapenem‐resistant pathogens.

In vitro antimicrobial susceptibility of Mycoplasma hominis genital isolates.

Sir, Mycoplasma hominis may be implicated in several diseases.[1] Data on the prevalence and the antimicrobial resistance of M. hominis from various countries are few and controversial.[1] We investigated the antimicrobial susceptibility of M. hominis from cervical and urethral swabs of outpatients in Northern and Southern Italy. A comparison of these data was done with similar studies worldwide, from 2011, to investigate the prevalence, therapeutic management and spread of antimicrobial resistance of this atypical pathogen. In two Italian hospitals of northern (Imola) and southern (Naples) Italy, a total of 2480 patients (1980 women and 500 men) aged 18–40, with cervicitis/ urethritis, were examined from July 2009 to December 2013. For each patient, two swabs from either the uterine cervix or urethra were collected and processed. The detection and the antimicrobial susceptibility testing (AST) of M. hominis genital isolates were performed using the Mycoplasma IST2 kit (bioMerieux, Marcy-l’Etoile, France). The techniques used for inoculation, diagnostic criteria and statistical analysis have been described earlier.[1] M. hominis was detected in 99 (4%) biological samples (84/1980 in women and 15/500 in men); significant bacterial load was revealed in 82 (3.3%). Colonization and infection by M. hominis decreased with increasing age (P < 0.001). AST was performed against all bacterial isolates. Tetracyclines exhibited a sensitivity percentage of 92.9%, streptogramins 96.0%, macrolides 28.5%, and fluoroquinolones 24.7% [Table 1].